The long-term goal of this research is to dissect the complex mechanism of regulation of virulence-associated gene expression in the pathogen Vibrio anguillarum. The plasmid-mediated iron uptake system is a major component of this bacterium?s virulence repertoire, playing an essential role in the host-bacteria interaction leading to systemic infection and mortality. This system serves as an excellent model for the study of the role that iron scavenging plays in systemic infections of humans since the pathogenesis of this infection shows striking similarities to human septicemic disease caused by other vibrios. We have characterized the essential components of this iron uptake system: the siderophore anguibactin, transport proteins, and regulatory elements controlling its synthesis and transport. In this system we identified an iron transport-biosynthesis operon whose expression is controlled by the positive regulators AngR and TAF that act at regions that include the promoter of this operon. We also characterized two antisense RNAs, RNAalpha and RNAbeta, that act post-transcriptionally in the negative control of the iron transport-biosynthesis operon. RNAalpha acts in the control of expression at the fatA-farB intergenic region and RNA beta acts as an attenuator of the expression of angR as compared to upstream genes in the operon. Therefore, we propose: (1) to characterize the roles of the two antisense RNAs, RNAalpha and RNA beta, as negative regulators of gene expression and to investigate the molecular and structural parameters that affect their stability, activity and influence on virulence. (2) To characterize the role of the positive regulators, AngR and TAFr, in the expression of the iron transport-biosynthesis operon, anguibactin production, and virulence. We intend to generate site-directed and deletion mutants in the angR gene and in the TAFr region in order to dissect the genes that are essential for regulation and virulence. The specific mechanisms by which these components govern the pathogen-host interaction will be further elucidated. Dissection of the molecular interactions underlying the expression of the iron uptake system in this pathogen will serve as a paradigm to the exploration of new avenues in understanding the process of systemic human infection of a bacterial etiology. In the long term, our findings will aid in the development of measures for the control of septicemic diseases.